11alpha-hydroxyprogesterone and esters thereof



Patented Jan. 12, 1954 Ila-HYDROXYPROGESTERONE AND ESTERS THEREOF Herbert 0. Murray and Durey H. Peterson, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan No Drawing. Application February 19, 1952, Serial No. 272,505

vascular disease as demonstrated in rabbits.

Moreover the product of the present invention does not exhibit undesirable side effects as does lls-hydroxyprogesterone such as, for example, catabolic efiects and anesthesia, progestational, glucocorticoid and androgenic activity and adverse effects on pregnancy as evidenced in rats.

Additionally, the esters have pharmacological and especially progestational and anti-estrogenic activity and are useful in the synthesis of other ll-oxygenated steroids such as cortisone.

The art is apprised of llp-hydroxyprogesterone, Shoppee and Reichstein, Helv. Chim. Acta, 24, 351-60(1941);.Reichstein and Fuchs, ibid., 26 684 (1943), melting at 182 to 188 degrees centigrade and with an [a] of plus 222.5 :4 degrees (1.748 in acetone). The novel compound of the present invention is distinguished therefrom in physical, chemical and physiological properties. The new compound, 1la-hydroxyprogesterone, melts at 166 to 168 degrees centigrade and has an hai of plus 180 degrees and a 76242 of 46.72. 11a. hydroxyprogesterone and '11s hydroxyprogesterone have specifically diilerent infrared absorption spectra. The lla-hydroxyprogesterone is thermally stable and resistant to attack by acids whereas llp-hydroxyprogesterone is unstable; lla-hydroxyprogesterone can be protected from oxidation whereas llfi-hydroxyprogesterone is unprotectable.

It is an object of the present invention to provide the novel lla-hydroxyprogesterone and esters thereof. Other objects will be apparent to those skilled in the art to which this invention pertains. s

The following examples are illustrative of the processes and products of the present invention but are not to be construed as limiting.

Example 1.-11 a-hydroxyprogesterone A medium was prepared fromfive milliliters of corn steep liquor, twenty grams of Edamine commercial lactalbumin digest and fifty milligrams of Cerelose commercial dextrose per liter of tap water and adjusted to a pH of between about 5.5 and 5.9. To four liters of this medium containing a 32 to 48 hour growth of culture RH 176, Rhizopus arrhizus, grown at room temperature with aeration, was added one gram of progesterone in fifty milliliters of acetone. The culture was then incubated at room temperature for 48 hours. At the end of thistime, thepH of the medium was 3.5 and the fermentation liquor and mycelia were extracted successively with three one-liter portions, one two-liter portion, and one one-liter portion of methylene chloride. The methylene chloride extracts were combined and washed with two loo-milliliter portions of a two percent aqueous sodium bicarbonate solution and three 500-milliliter portions of distilled water. The methylene chloride extract was evaporated to dryness in vacuo and the solid residue taken up in fifty milliliters of methylene chloride. The solution was transferred to a IOU-milliliter beaker and evaporated by a stream of air. The solids, weighing 1.585 grams, were dissolved in five milliliters of hot methanol and allowed to cool slowly to room temperature. Seventy-five milligrams of crystals, melting at 246 to 249 degrees centigrade, separated from the mother liquor.

The mother liquor from the above crystallizetion was freed of solvent by aeration and the solid residue weighing 1.5 grams was dissolved in fifty milliliters of benzene, and chromatographed over alumina (A1203) as shown in Table I. Fifty grams of acid-washed alumina, dried at degrees centigrade, was used as adsorbent and loo-milliliter portions of solvents were used to develop the column (see Table I).

TABLE I A1203 chromatography of biologically converted progesterone using organism RH 176 (Rhizopus arrhizus Eluate Solvent Solids,

Milligrams benzene 0 do 182.2 benzene plus 5 percent ether 123.0 do 246. 6 162. 9 18.0 11.2 128. 2 33. 6 16.1 15. 4 3.8 2.0 3. 4 3. 6 3. 5 2.1 2. 5 222. 6 167. 9 23. 5 17. 3 B. 7 30. 9 6. 1 13. 0 l o 8. 4 acetgne plus 5 percent methano 25.0 o 4. 6 acetone plus 10 percent methanol 7. 4 31 do. 1.4 32- acetone plus percent methanol S. 1 33 ..do 5. 3

3 Combined @eluate solids fractions 19 and 20 were dissolved in two milliliters of hot methanol and filtered. After overnight refrigeration, 171 milligrams of crystalline product, melting at 116.6

to 167 degrees centigrade, was obtained. Eecrystallization from three milliliters ofmethanolgprozmilligrams of chromic oxide in five microliters sivelywith It'WO tone-half oywolmne portions of of water and allowing the .mixture to stand at A comparison :of the physical constants vindi- I cated that the triketone was i'identical 110 the known 311-1 -ketoprogesterone, melting at .172 to 1'75 :degreescentigrade; .lal ;of :plus:238 degrees plus or minus eight degrees (0:91in acetone). Infrared analysis, X-ray diffraction, paper chromatography and optical z-rotationall showed thatthe compound was identical to -an authentic sample of ll-ketoprogesterone. Reichstein, Helv. Chim. Acta, 23, 684 (1940); ibid..-26, 12111942).

Example 2.-1;la-'lrydroa:ypr0gesterone A medium was prepared :of twenty grams .of Edamine enzymatic digest of lactailbumin, three grams of corn steep liquor and fifty'grams of technical dextrose diluted to one liter "with tap water and adjusted to a pH of 4.3 to 4.5. Twelve liters of this sterilized medium was inoculated with .Rhiaopus nigmcans minus strain, .American type culture collection number 6227b, and incubated for 24 hours at a temperature of 28 degrees centigrade using a rate of aeration and stirringzsuch that the :oxygen uptake was 6.3 to .7 millimoles per hour per liter of jNazSOa according to the method of CoopenFernstrom-and Miller, Ind. Eng. Chem. 36, 504. (1944). To this medium containing a .24 hour growth of Rhizomes nigricans minus strain was added six grams of progesterone in 150 milliliters of acetone to proside a suspension-of 'the steroid in the culture. .After an additional 24 hour period .of incubation under the same conditions of temperature and aeration, the beer and mycelium were extracted and concentrated. The mycelium was filtered, washed twice, each time with a volume of acetone approximately equal to the .volume .of the mycelium-and extracted twice, each time with a volume of .methylene chloride approximately equal to thevolumeofthe-mycelium. The acetone and methylene chloride extracts including sol-- vent were-added to the beer filtrate. 'The mixed extracts and beer filtrate were extracted succes- .methylene chloriide and'gthen with two mic-fourth by volume portions of methylene chloride. The combined methylene chloride extracts were washed with two one-tenth by volume portions 0! a two ;percent aqueous solution of sodium bicarbonate and then with two one-tenth by volume portions of water. .After drying the methylene chloride :extractswith about three to five grams of anhydrous sodium sulfate per liter of solvent and filtering, the *solvent was removed by distillation. fI heresidue was dissolved in a minimum :01" :methylene chloride, filtered and the solvent then evaporated. The resulting crude crystals were dried and then washed five times with five milliliter portions of tether per gram of crude .crystalsgyielding ;5;.072 gram of crystals melting at to 168 degrees centigrade. Recrystallization :of .400 :milligrams :of ithese icrystals ifrom methanol gave .311 milligrams of "l lu-hydroxy- .progesteron'e witha melting iapoin't :of 166 to 168 degrees centigrade,:an iul :of plus 1.80 :degrees (-1-.0127 inzchloroform) andakzlz'of r4672.

l lnalysis: ".Galculated for .C2IH30O32 C, *LYZBAO; 'I-l,19.-10. FoundziLflfifl'l; 31118.92.

Example ,3relic:acetoxypxogestemne Twenty milligram of tl'lcehydroxyprogesterone was :mixed with 0.6 milliliter 10f pyridine :and 0.6 milliliter of acetic anhydride. After sixteen hours:atroom -.temperature,125:milliliters of water wasadded. .After one hour, the preparation was refrigerated to cause crystallization. I'h'eicrystals which formed were washed with :water and driedto yield 116.1 zmilligrams of i-l'laeacetoxyprogesterone .having a Jnelting zpoint iOf .to 177 degrees centigrade, [c1 3 -:of 1143 .degrees inacctone andplus I518 degrees linichloroform extinction ,coeffic'ieritikzelzin IQIGQ hOIiOf 43.44, 3E:16,=1 75. Infrared analysis indicated the absence of the hydroxyl group and the presence of a new :ace- .toxygroup. V Anaylsis: Ga'lculated ior anetoxyprogesterone: :G, 751),; .H, 8;85. .Found': 57433; H,.8fl8.

Example 4.-11a fiormylowmogesterone :A .solution .aof :fiveigramsaof I-l w-hydroxyproges- -terone"-in;l00milliliters .of 8'7wpercent formic acid was :heated rtO 3'15 degrees -.centigrade for ninety minutes. The :solution :was then cooled to :degrees :centigrade :and 140 :milliliters of water :was added to initiate crystallization. additional 35 milliliters of water was then added and the mixture 'placedzin the refrigerator. The yield was 3.29 :grams .658 percent yield) of crystals imelting .at 15.4 fto 15.8 degrees :centigrade. The mother liquor was diluted with water to about 400 milliliters and allowed to stand for two-days under refrigeration ;at :a :temperature .0f four 'degreescentigrade, to give a second :crop of crystals weighing {0.163 gram deleven percentwild') ng at 152 to .155 degrees centigrade. The two crops were combined and recrystallized from 139-:milliliters of :methanol yielding crystals of Ila-:Ioimyloxyprogesterone'melting at 157 -to -15!) degrees :centigrade. :iliil of plus i l? degrees -c01752 gram in 1'100mi1liliters er chloroform).

Example.5.--1,1a-formyloxyprqgeszer 'One 'gramof 1 la-hydroxyprogesterone "in twen- :ty milliliters of 87 percent 'form'ic acid was heated for 'two ;hours at "-75 :degrees -centigrade. aUpon incorporatmg 200 milliliters of waterpthere zprecipitatedsO-ifi gram of crystals melting at 1 52 $0154 degreescentigrade. "Two recrystallizations from forty-milliliter portions of 45 percent formic acid gave 0.64 gram (56 percent yield) of 11aformyloxyprogesterone melting at 158.5 to- 161.5 degrees centigrade.

Example 6.-11 a-propi0nyloxyprogesterone cent of the theoretical yield. The lla-propionyloxyprogesterone had a melting point of 145.5 to 146 degrees centigrade (uncorrected) and an optical rotation [a] of plus 156 degrees (1.253 rams in chloroform).

Analysis: Calculated for C24H34O42 H, 8.87. Found: C, 74.84; H, 8.74; C, 8.64.

Example 7.-11a-benzoxyprogesterone lla-hydroxyprogesterone (0.5 gram) was dissolved in six milliliters of hot benzene and cooled to room temperature. Then 0.4 milliliter of freshly dried and redistilled pyridine and 0.4 milliliter of freshly-distilled benzoyl chloride were added and the mixture allowed to stand at room temperature for twenty hours. The reaction mixture was diluted with fifty milliliters of ether, washed successively with water, ten percent sodium hydroxide solution, and water, dried over sodium sulfate and the solvent evaporated on a hot water bath, the terminal evaporation being facilitated by vacuum. The residue was heated with 25 milliliters of water under reflux for fifteen minutes, cooled, extracted with ether,

washed with water, ten percent sodium hydroxide solution, and again with water, dried and evaporated to a volume of ten milliliters. Crystals formed during this evaporation. After cooling, the crystals were removed by filtration and washed with about five milliliters of hexane. The yield was 0.44 gram (68 percent of the theoretical) of lla-benzoxyprogesterone melting at 179 to 181 degrees centigrade. Recrystallization from twenty milliliters of ether concentrated on a steam bath to ten milliliters gave a product melting at 181 to 183 degrees centigrade; [0.1 of plus 88 degrees (1.686 grams in chloroform).

Analysis: Calculated for C2eH34O4Z C, 77.38; H, 7.89. Found: C, 77.50; H, 7.88; C, 77.64; H, 8.01.

The lla-hydroxyprogesterone can be converted into the corresponding lla-acyloxy derivatives as indicated in Example 3, according to known acylation procedures, as by reaction with ketene, :etenes or an appropriate acid, acid anhydride or acyl halide, in an organic solvent such as pyridine or the like. Representative esters of lla-hydroxyprogesterone thus-prepared include one to eight carbon atom carboxylic acid acyloxy esters of saturated or unsaturated aliphatic or cycloaliphatic, aryl, alkaryl, aralkyl, mono, di or polycarboxylic acids, formyloxy, acetoxy, propionyloxy, butyryloxy, valeryloxy, hexanoyloxy, heptanoyloxy, octanoyloxy, benzoxy, phenylacetoxy, toluoyloxy, cyclopentanoyloxy, cyclopentylpropionyloxy, acrylyloxy, cyclohexanoyloxy, the half and di esters of the lla-hydroxyprogesterone with malonio, maleic, succinic, glutaric and adipic acids, and the like. The acyl groups may also contain non-interfering substituents, such as mono or poly, halo, chloro, bromo, hydroxy, methoxy or the like if desired.

This application is a continuation-in-part of the applications of Murray and Peterson, Serial Number 180,496, filed August 19, 1950, now abandoned, and Serial Number 264,640, filed January 2, 1952.

We claim:

1. lla-hydroxyprogesterone.

2. A compound represented by the graphical formula:

terone.

HERBERT C. MURRAY. DUREY H. PETERSON.

References Cited in the file of this patent Reichstein, Helv. Chim. Acta 23, 684-8 (1940). 

1. 11A-HYDROCYPROGESTERONE. 